Project Summary BCG is a potent stimulator of innate immunity and has been used as an adjuvant in cancer therapy. BCG has also been shown to improve TB-unrelated infant morbidity and mortality and improve infant responses to vaccines in countries where BCG is routinely administered to neonates. However, epidemiologic studies have generated conflicting results due to their design and/or to the different BCG products. Here we propose to study the nonspecific protective effect of BCG by investigating innate and adaptive immune responses to infection with the rotavirus live attenuated vaccine virus (RoV) as a surrogate of infection. We will measure responses in Brazilian infants, who receive BCG in the first week of life per standard of care (BCG+), and in US infants, who do not receive BCG (BCG-). We will also investigate the mechanism of BCG's nonspecific protection against heterologous infections. Our results may create a framework for immune modulatory interventions in infants, who have higher infectious morbidity and mortality and lower responses to vaccines compared with older children and young adults. Moreover, we will investigate the effect of BCG both in HIV- exposed uninfected (HEU) and in HIV-unexposed (HUU) infants. HEU are a growing population with increased susceptibility to infections, hospitalization and death and with multiple innate and adaptive immune defects compared with HUU, which makes them important candidates for boosting immune interventions. We hypothesized that the protective effect of BCG against heterologous infections in infants is mediated by activation of trained innate immunity that controls the initial stages of infection and supports generation of robust adaptive immune responses.This hypothesis will be addressed in the following Specific Aims: Aim 1. To assess the magnitude of innate and adaptive immune responses to RoV and their association with viral clearance in BCG+ HEU and HUU in comparison with BCG- HEU and HUU. We will measure innate immune cell phenotypes, cytokine expression, RoV-specific humoral and cellular immunity, and RoV shedding, after administration of the RoV vaccine at 6 weeks of age. Aim 2. To investigate the functional and epigenetic effect of BCG on innate immunity and develop an in vitro system that reproduces this effect using BCG and pattern recognition receptor (PRR) agonists. We will develop an in vitro system of BCG stimulation that reproduces the NK, ???T and antigen presenting cell phenotypic and DNA methylation profiles observed after in vivo BCG administration. Then we will develop a cocktail of PRR agonists that reproduces the effect of in vitro BCG stimulation and could be translated for clinical use as an immune boosting agent for HEU and other high risk infants. Impact. Using objective laboratory-based outcome measures and an ample sample size, this study will provide the highly needed definitive proof of the beneficial effect of BCG heterologous immune stimulation in infants.